The present invention relates a novel xcex1-amylase within the family of Termamyl-like xcex1-amylases suitable for detergents. The invention also relates to variants (mutants) of parent Termamyl-like xcex1-amylases, notably variants exhibiting increased thermostability at acidic pH and/or at low Ca2+ concentrations (relative to the parent) which are advantageous with respect to applications of the variants in, industrial starch processing particularly (e.g., starch liquefaction or saccharification). Said xcex1-amylase and xcex1-amylase variants of the invention may advantageously also be used in detergents.
xcex1-Amylases (xcex1-1,4-glucan-4-glucanohydrolases, EC 3.2.1.1) constitute a group of enzymes which catalyze hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides.
There is a very extensive body of patent and scientific literature relating to this industrially very important class of enzymes. A number of xcex1-amylase such as Termamyl-like xcex1-amylases variants are known from, e.g., WO 90/11352, WO 95/10603, WO 95/26397, WO 96/23873 and WO 96/23874.
Among more recent disclosures relating to xcex1-amylases, WO 96/23874 provides three-dimensional, X-ray crystal structural data for a Termamyl-like xcex1-amylase which consists of the 300 N-terminal amino acid residues of the B. amyloliquefaciens xcex1-amylase and amino acids 301-483 of the C-terminal end of the B. licheniformis xcex1-amylase comprising the amino acid sequence (the latter being available commercially under the tradename Termamyl(trademark)), and which is thus closely related to the industrially important Bacillus xcex1-amylases (which in the present context are embraced within the meaning of the term xe2x80x9cTermamyl-like xcex1-amylasesxe2x80x9d, and which include, inter alia, the B. licheniformis, B. amyloliquefaciens and B. stearothermophilus xcex1-amylases). WO 96/23874 further describes methodology for designing, on the basis of an analysis of the structure of a parent Termamyl-like xcex1-amylase, variants of the parent Termamyl-like xcex1-amylase which exhibit altered properties relative to the parent.
WO 95/35382 (Gist Brocades B. V.) concerns amylolytic enzymes derived from B. licheniformis with improved properties allowing reduction of the Ca2+ concentration under application without a loss of performance of the enzyme. The amylolytic enzyme comprises one or more amino acid changes at positions selected from the group of 104, 128, 187, 188 of the B. licheniformis xcex1-amylase sequence.
WO 96/23873 (Novo Nordisk) discloses Termamyl-like xcex1-amylase variants which have increased thermostability obtained by pairwise deletion in the region R181*, G182*, T183* and G184* of the sequence shown in SEQ ID NO: 1 herein.
WO 97/00324 (KAO) disclose a gene encoding an alkaline liquefying xcex1-amylase derived from Bacillus sp. strain KSM-AP1378 with the deposited no. FERM BP-3048 suitable for detergents.
The present invention relates to a novel xcex1-amylase and to novel xcex1-amylolytic variants (mutants) of a Termamyl-like xcex1-amylase, in particular variants exhibiting increased thermostability (relative to the parent) which are advantageous in connection with the industrial processing of starch (starch liquefaction, saccharification and the like). The novel xcex1-amylase is suitable for laundry washing and dishwash as is has a high activity under wash conditions at alkaline pHs in the range 9-11.
The inventors have surprisingly found out that in case of combining two, three, four, five or six mutations (will be described below), the thermostability of Termamyl-like xcex1-amylases is increased at acidic pH and/or at low Ca2+ concentration in comparison to single mutations, such as the mutation dislcosed in WO 96/23873 (Novo Nordisk), i.e., pairwise deletion in the region R181*, G182*, T183* and G184* of the sequence shown in SEQ ID NO: 1 herein.
The invention further relates to DNA constructs encoding variants of the invention, to composition comprising variants of the invention, to methods for preparing variants of the invention, and to the use of variants and compositions of the invention, alone or in combination with other xcex1-amylolytic enzymes, in various industrial processes, e.g., starch liquefaction.